کد مقاله | کد نشریه | سال انتشار | مقاله انگلیسی | نسخه تمام متن |
---|---|---|---|---|
2020168 | 1542317 | 2016 | 9 صفحه PDF | دانلود رایگان |
عنوان انگلیسی مقاله ISI
A simplified protocol for high-yield expression and purification of bacterial topoisomerase I
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کلمات کلیدی
IPTGIMACTopoisomerase IAutoinductionVGStris-acetate-EDTANi2+-NTAOTGviridans group streptococci - streptococci گروه viridansStreptococcus mutans - استرپتوکوک موتانسCharacterization - تعیین مشخصاتTAE - توnickel-nitrilotriacetic acid - نیکل نیتریلوتوریکات اسیدPurification - پاکسازیImmobilized metal-ion affinity chromatography - کروماتوگرافی وابسته به فلز فلز یونی
موضوعات مرتبط
علوم زیستی و بیوفناوری
بیوشیمی، ژنتیک و زیست شناسی مولکولی
زیست شیمی
پیش نمایش صفحه اول مقاله
چکیده انگلیسی
Type IA topoisomerases represent promising antibacterial drug targets. Data exists suggesting that the two bacterial type IA topoisomerase enzymes-topoisomerase I and topoisomerase III-share an overlapping biological role. Furthermore, topoisomerase I has been shown to be essential for the survival of certain organisms lacking topoisomerase III. With this in mind, it is plausible that topoisomerase I may represent a potential target for selective antibacterial drug development. As many reported bacterial topoisomerase I purification protocols have either suffered from relatively low yield, numerous steps, or a simple failure to report target protein yield altogether, a high-yield and high-purity bacterial topoisomerase I expression and purification protocol is highly desirable. The goal of this study was therefore to optimize the expression and purification of topoisomerase I from Streptococcus mutans, a clinically relevant organism that plays a significant role in oral and extra-oral infection, in order to quickly and easily attain the requisite quantities of pure target enzyme suitable for use in assay development, compound library screening, and carrying out further structural and biochemical characterization analyses. Herein we report the systematic implementation and analysis of various expression and purification techniques leading to the development and optimization of a rapid and straightforward protocol for the auto-induced expression and two-step, affinity tag purification of Streptococcus mutans topoisomerase I yielding >20Â mg/L of enzyme at over 95% purity.
ناشر
Database: Elsevier - ScienceDirect (ساینس دایرکت)
Journal: Protein Expression and Purification - Volume 124, August 2016, Pages 32-40
Journal: Protein Expression and Purification - Volume 124, August 2016, Pages 32-40
نویسندگان
Jesse A. Jones, Emily Price, Donovan Miller, Kirk E. Hevener,